Recently, Adeno-Associated Virus (AAV)-based vectors have emerged as promising gene delivery vehicles for a wide array of diseases, including cardiovascular disorders. Despite early encouraging results, the CUPID (Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease) trial using an AAV serotype 1 vector encoding the sarcoplasmatic calcium ATPase SERCA2a failed to meet both its primary and secondary endpoints. These results were surprising because in porcine models of HF AAV1.SERCA2a improved cardiac function. Preliminary results suggest that the disappointing outcome was due to a failure of AAV1.SERCA2a to deliver efficiently the SERCA2a gene. One possible explanation for the poor gene delivery is that neutralizing antibodies (NAbs) against AAV1 were not detected with the in vitro NAb assay used in the CUPID trial, but that these NAbs prevented transduction. In Aim 1 of this application we will test in a porcine HF model the hypothesis that extremely low levels of NAbs, which can only be detected by a more sensitive in vivo NAb assay, can prevent transduction and therapeutic efficacy of AAV1.SERCA2a. Conversely, if in vitro NAb assays are sufficiently sensitive, we will determine the maximal NAb levels that are still compatible with efficient transduction and therapeutic efficacy of AAV1.SERCA2a (in pigs). An alternative explanation for the negative results of the CUPID trial is that AAV1 displays specie-specific tropism, i.e. that AAV1 can efficiently transduce pig but not human cardiomyocytes. To bring cardiac AAV gene therapy to the clinic, it will be critical to isolate AAV variants that 1) Can efficiently transduce human cardiac cells and 2) Show increased resistance to NAbs. The isolation of such variants is the goal of Aim 2. Unfortunately, it seems unlikely that AAV variants that can efficiently transduce human cardiomyocytes and that are also resistant to very high levels of NAbs against the naturally occurring AAV serotypes can be isolated. Therefore, in Aim 3, we will test an approach to deplete NAbs from the blood by plasmapheresis coupled with immunadsorption with columns with immobilized AAV particles. With the successful completion of this proposal, we will have established whether an in vitro NAb assay is sensitive enough to serve as an exclusion criterion for cardiac AAV gene therapy trials where AAV is delivered by intracoronary infusion, or if a more sensitive in vivo assay must be used. We will have isolated novel AAV variants with tropism for human cardiomyocytes and increased resistance to NAbs. Finally, we will have established whether plasmapheresis coupled with immunadsorption on AAV columns can be used to deplete NAbs from blood. These parameters will be critical in the design and execution of future gene therapy trials for cardiovascular diseases.